Glycolipid complexes and their uses

ABSTRACT

A complex between a water-soluble polymer containing hydrophobic regions and a glycolipid, immunoassays utilizing the complex as an immune reagent and diagnostic methods to be used in the context of neuropathy and insulin-dependent diabetes. The diagnostic methods employ anti-glycolipid antibody as a marker. The preferred glycolipid is sulfatide.

TECHNICAL FIELD

The present invention relates to novel forms of glycolipids and novelmethods for assaying anti-glycolipid antibodies. The preferredglycolipid is sulphatide. The novel assaying methods are particularlyadapted for the diagnosis of any disease for which elevated levels of ananti-glycolipid autoantibody is a marker. The disease may be, forinstance, neuropathy and insulin-dependent diabetes (IDDM, Type 1diabetes) including diagnoses excluding Type 2 diabetes, latecomplications associated therewith and monitoring the treatment of IDDM.The novel forms of sulphatide may potentially find use as a medicamentin the treatment of IDDM.

BACKGROUND OF THE INVENTION

Sulphatide, the most important antigen of the present invention is aglycosphingolipid having the structure:

(=galactosylceramide-3-sulphate).

The development of manifested IDDM involves an autoimmune process wherethe insulin producing cells in the islet of Langerhan in the pancreasgradually are knocked out. At the time of diagnoses 70-80% of the cellshave been destroyed and the remaining cells will generally disappearwithin a 5 year period. Prediabetic individuals are those which have anongoing destruction of insulin-producing cells but still withoutclinical symptoms. A particular type of prediabetic individuals arenon-IDDM patients whose disease will switch to IDDM.

In the context of the invention the term IDDM include manifested IDDM aswell as preforms thereof, if not otherwise specified.

The main marker autoantibodies found in IDDM are islet cell antibodies(ICA), anti-glutamic acid decarboxylase antibodies (anti-GAD),anti-insulin antibodies (IAA), antibodies against 37 kD protein (likelythe same as tyrosine phosphatase) and anti-sulphatide antibodies. Themain disadvantages of these markers are:

ICAs are determined by conventional immunohistochemistry which make themunsuitable for large screenings. They are normally considered goodpredictors of IDDM but their assay suffers from relatively high intra-and interassay variations.

Anti-GAD antibodies are easy to determine also in large scalescreenings, but they may not be of a strong predictive value.

IAAs are fairly easy to determine but are not present in more than40-50% of newly diagnosed type 1 diabetic children and is less common inadult patients.

Anti-37 kD protein antibodies have a significant lower frequency (about50%) than ICA and anti-GAD antibodies.

Both ICAs and anti-GAD antibodies have been used for the diagnoses ofprediabetics.

Anti-sulphatide antibodies have been recognized as a good marker formanifested IDDM (Buschard et al., Lancet 342 (1993) page 840-; andBuschard et al., APMIS 101 (1993) 963-970). Results supporting adiagnostic link between this type of antibodies and prediabetic forms ofIDDM have not hitherto been published. Increased titers ofanti-sulphatide antibody have been found in neuropathy patients andcould be used for identification of individuals that might developneuropathy. Part of diabetes type 2 patients also develop neuropathy. Ina preliminary study one of the present inventors has shown that diabetestype 2 patients without neuropathy did not have anti-sulphatide antibodyreactivity and there is thus a possibility that those with neuropathydevelopment will be positive and thus anti-sulphatide antibody might beuseful as a prognostic tool. See Fredman et al., J. Neurolog. 238 (1991)75-79.

Serum levels of antibodies towards glycosphingolipids have often beenassayed by a combination of thin layer chromatography (TLC) of theantigens and ELISA methodology (enzyme-linked immuno sorbent assay)employing the chromatographed material as antigen for assayingoccurrence of serum antibodies (TLC-ELISA) (Fredman et al., J. Neurol.238 (1991) 75-79; and Fredman et al., J. Neurol. 240 (1993) 381-387).

The assaying methods of anti-glycolipid antibodies have beenproblematic, in particular the methods for anti-sulphatide antibodies.

Sulphated glycolipids, in particular sulphatide,lactosylceramide-3-sulphate and seminolipid and corresponding antibodieshave been suggested as diagnostic markers and therapeutic agents in thecontext of diabetes (Buschard K, WO-A-9219633).

We have tried to vary the attachment of sulphatide to plastic wells, andhave clear evidences that direct adsorption results in a highcoefficient of variation and often gives unspecific reactions withoutany correlation to earlier TLC-ELISA findings. The criticality ofexposing correctly both the lipid moiety and the carbohydrate moiety ofshort carbohydrate chain glycolipids has also been illustrated withmonoclonal anti-sulphatide antibodies. Thus, the monoclonalanti-sulphatide antibody described by Fredman et al (Biochem. J. 251(1988) 17-22) is sensitive to changes in the lipid part. Another aspectis that galactose substituted with sulphate is a common epitope onseveral glycoproteins and glycolipids, and there are no results so farsupporting that a general reactivity towards this epitope is relevantfor the development of IDDM and its late complications. Accordingly, themain problem the invention sets out to solve relates to the presentationof glycolipid epitopes.

Another problem the invention sets out to solve is connected to thediagnoses of prediabetic forms of IDDM.

SUMMARY OF THE INVENTION

A first objective of the invention is to improve the presentation ofglycolipid antigens/haptens in immunoassays and potentially also intherapy. The antigens/haptens primarily concerned exhibit shortcarbohydrate chains.

A second objective is to provide improved immunoassays for measuringanti-glycolipid antibodies or glycolipid antigens/haptens, in particularautoantibodies against glycosphingolipid antigens exhibiting mono-and/or disaccharide units that may be sulphated.

A third objective is to provide improved diagnostic methods utilizing asmarkers auto-antibodies binding to one or more of the previouslymentioned glycolipid antigens/haptens for diagnosing IDDM includingpreforms thereof.

A fourth objective is to provide diagnostic methods for determiningpreforms of IDDM.

These objectives may be complied with by complexing the glycolipidantigens/haptens as defined above to a polymeric hydrophilic carrierexhibiting hydrophobic regions. Examples of suitable carrier moleculesare delipidized forms of hydrophilic proteins, such as albumin, that arecapable of associating to lipid compounds, such as fatty acids andderivatives thereof.

DETAILED DESCRIPTION

Accordingly a first aspect of the invention is a complex between awater-soluble carrier polymer and a glycolipid antigen, preferably aglycosphingolipid antigen, that exhibits a mono- or disaccharide unitthat may or may not be sulphated in the 3-position of its terminalcarbohydrate unit. The water-soluble polymer is preferably a delipidizedform of a protein that have hydrophobic regions that are capable ofbinding to lipids, such as fatty acids. At the priority date the mostpreferred water-soluble polymer was delipidized albumin. Thewater-soluble polymer may be insolubilized by covalent attachment orphysical adsorption to any of the known solid phases used inimmunoassays or chromatography. Insolubilization may be carried outeither before or after complex formation with the glycolipidantigen/hapten concerned. In this aspect of the invention the best modeencompasses sulphatide as the glycolipid antigen/ hapten. The forceskeeping the glycolipid antigen/hapten complexed is believed to be mainlyhydrophobic, meaning that at the priority the preferred variant of theinventive complex is thought to be a non-covalently associated complexbetween the carrier polymer and the glycolipid.

A second aspect of the invention is an immunoassay for anti-glycolipidantibodies or glycolipid antigen/hapten utilizing the above-mentionedglycolipid complex as an antigen. In this type of assays, an immunecomplex is formed between the inventive type of glycolipid-carrierpolymer complex and the appropriate anti-glycolipid antibody (analyte)of a sample in an amount that is related, either qualitatively orquantitatively, to the amount of anti-glycolipid antibody in the sample.In an alternative embodiment the glycolipid antigen/hapten is theanalyte which is allowed to compete with the glycolipid-carrier polymercomplex as defined above for binding to an added anti-glycolipidantibody. In order to facilitate measurement, further reactants that arecapable of being incorporated into the immune complex may be included,e.g. labelled reactants (labelled antibodies or labelled antigens) orinsoluble or insolubilizable reactants. Examples of labels that can beused are enzymes, enzyme substrates, cofactors, coenzymes, fluorophores,dyes, particles such as latex particles, carbon particles, metalparticles, radioactive isotopes etc. The labelled reactant is used in anamount so that the amount incorporated and/or not incorporated into the(glycolipid) - - - (anti-glycolipid antibody) complex becomes a measureof the level of analyte in the sample. In case the signal from the labelchanges upon being incorporated into the immune complex, no physicalseparation of complex-bound from non-complex-bound form of the label isnecessary before measuring the signal from the label. In case complexformation does not lead to any signal change, physical separation ofcomplex-bound from non-complex-bound form of the label becomesimperative. In case separation is accomplished one speaks aboutheterogeneous assays while otherwise the assays are called homogeneous.Physical separation of label not incorporated into the immune complexfrom label incorporated into the immune complex is normally accomplishedby utilizing a reactant that is insoluble or insolubilizable in theassay medium.

Other ways of subdividing immunoassays are in competitive andnon-competitive assays (sandwich). Still other types are agglutinationassays, turbidometric assays, precipitation assays that may or may notbe homogeneous/heterogeneous and/or competitive/non-competitive etc.

The normal conditions for immune assays are applicable, which means thatthe pH during immune reactions normally is within the range 4-11, thetemperature between 0-35° C. etc. For heterogeneous assays, each of thevarious antigen-antibody reactions contemplated are normally followed byintermediate separation and washing steps to remove non-specificallybound immune reactants and other disturbing substances. The medium forthe reactions is normally water (aqueous) buffered to the appropriatepH.

The sample used derives from a body fluid and may be a blood sample suchas whole blood, serum or plasma, or any other type of sample that maycontain the anti-glycolipid antibody or glycolipid antigen/haptenconcerned (urine, cerebrospinal fluid, lacrymal fluid, saliva etc.)

The immunoassays of this aspect of the invention may be used either fordiagnoses or in order to screen for or characterize monoclonalantibodies directed towards glycolipid antigens of the above-mentionedtype.

As per the priority date the most preferred mode of the second aspect ofthe invention is given in the experimental part and encompassesdelipidized albumin as the carrier polymer fixed to a solid phase andsulphatide as the glycolipid. The assay protocol encompasses a threelayered sandwich assay in which the sample is incubated with the carrierpolymer which subsequently is incubated with labelled anti-humanantibody to the formation of the insolubilized ternary immune complex:glycolipid - - - anti-glycolipid antibody - - - labelled anti-antibody.

The third aspect of the invention is a method for diagnosing disordersrelated to an elevated level of anti-glycolipid antigen/hapten antibodyas defined above by utilizing an immunoassay of the above-mentionedtype. With respect to anti-sulphatide antibodies the method may beapplied in the context of diagnosing neuropathy or IDDM includingprediabetic forms thereof and monitoring of treatment regimens, such asprophylactic treatment e.g. with insulin. Uses included are differentialdiagnoses of IDDM/non-IDDM and/or diagnoses/prediction of diabetic latecomplications (neuropathy, retinopathy, and neuropathy) and/ormonitoring of treatment regimens in general. A potential importantdiagnostic use included is the determination of preforms of IDDM innon-IDDM individuals.

A fourth aspect of the invention relates particularly to the diagnosesof prediabetic forms of IDDM utilizing elevated sample levels ofanti-sulphatide antibody as a marker for a prediabetic state in thepatient from which the sample derives. In this aspect any type of assay,particularly immunoassays, may be used, although the best results will,with the present knowledge, be obtained in case the assay is run inaccordance with the above-mentioned third aspect with anti-sulphatideantibody as the analyte.

It has been realized during the development of the present inventionthat the diagnostic use of the inventive immunoassay method for elevatedlevels of anti-sulphatide antibody may be further improved in case italso takes into account elevated levels of other marker autoantibodiesfound in the context of IDDM as described above, for instance islet cellantibodies (ICA), anti-glutamic acid decarboxylase antibodies(anti-GAD), anti-insulin antibodies (IAA), antibodies against 37 kDprotein (likely the same as tyrosine phosphatase). It is believed thatin particular a simultaneous finding of elevated levels ofanti-sulphatide antibody and anti-GAD improves the diagnostic value byincreasing the specificity.

A potent fifth aspect of the invention, is to use the complex defined inthe context of the first aspect of the invention as the activeingredient/drug in pharmaceutical compositions. The preferred modeincludes the above-mentioned novel sulphatide complexes to preventand/or delay development of diabetes (IDDM) and to the treatment ofdiseases related to late complications of diabetes (IDDM). The complexesconcerned may, for example, interact with the immunological ligandand/or immune active cells involved in the pathogenic process andthereby inhibit their binding to glycolipid/sulphatide in target cellslike α- and β-cells of the pancreas. Potentially the glycolipidcomplexes of the first aspect of the invention may also interactpharmacologically in intracellular events involved in the diseaseprocess and caused by sulphatide and/or its metabolic products. Inparticular sulphatide complexes as defined above may be used as vaccinesin order to prevent, delay or alter IDDM or its late complications.

During the priority year it has been realized that diagnoses in thecontext of IDDM by assaying for anti-sulphatide antibodies may befurther improved in case one also accounts for elevated levels ofanti-GAD antibody. The improvement mainly relates to an increased in thespecificity. This type of diagnoses is not linked to any specific methodfor assaying anti-sulphatide or anti-GAD antibodies, although at thefiling date it was preferred to utilize the novel forms of glycolipidsas described for assaying anti-sulphatide antibody. Employment ofelevated levels of both anti-sulphatide and anti-GAD antibodies incombination as markers in the context of IDDM as described above is aseparate invention.

EXPERIMENTAL PART

Production of Delipidizid Albumin

Albumin was delipidized with hexane containing 5% glacial acetic acid,at +4° C. After thorough washing with hexane the albumin was dialyzedagainst Milli Q-water and lyophilized.

Biotinylated Delipidized Albumin

Fatty acid free albumin as described above was dissolved in 0.1 M NaHCO₃to a concentration of 10 g/L. Biotin-N-hydroxy succinimide, 0.1 M indimethyl formamide, was added to a final concentration of 0.02 M. Thefinal mixture was left at 20° C. for 60 min and an equal volume ofphosphate buffered saline (PBS) was added. The reaction product wasdialysed against 5 changes of PBS at +4° C. for 24 hs.

Preparation of Sulphatide

Bovine brain was homogenized with an equal volume of water. Methanol andchloroform were then added to a final ratio of 4:8:3 (by volume,chloroform-methanol-water). The lipid extract obtained was freed fromparticles by centrifugation. Low molecular weight components wereremoved by partition after addition of chloroform and methanol to aratio of 4:2:1 (by volume, chloroform-methanol-water). The crude lipidextract was chromatographed on silica gel. The isolated sulphatidefraction was saponified and rechromatographed on silica gel. Afterrepeated partition the sulphatide fraction was further purified byion-exchange chromatography. Finally the purified fraction was dissolvedin methanol and precipitated by addition of acetone. The isolatedsulphatide fraction was characterized by mass spectrometry (FAB-MS). Therecovery was about 1.5 g sulphatide per kg of bovine brain.

Adsorption of Sulphatide to Albumin

Sulphatide (100 nmole in chloroform-methanol-water mixture wasevaporated to dryness and redissolved in 500 μl sodium acetate (0.05 MpH 4.5) and sonicated for 15 min at room temperature. To this solutionwas added 0,5 mL delipidized albumin dissolved in the same buffer (2mg/mL). The mixture was incubated over night at room temperature undercareful mixing. Thereafter the albumin with sulphatide adsorbed wasprecipitated by the addition of 50 μL of 10% TCA (trichloro acetic acid)in water at +4° C. After 30 min at +4° C. the mixture was centrifuged(+4° C.) at 23,000×g for 3 min. The pellet was suspended in 1 mL PBS andkept at +4° C. The procedure could also be applied to biotinylatedalbumin.

Binding Sulphatide-albumin to a Solid Phase

Sulphatide-albumin was suspended in 50 mM carbonate buffer pH 9.6 to aconcentration of 2.5 mmole/mL and 100 μL of this solution was added tothe wells of a microtiter plate (Nunc Storwell Maxisorp immunomoduler,Denmark). The plate was incubated for more than 2 hours at 37° C. andwas then sealed with parafilm and kept at +4° C. until used. Just priorto the assay, the coating solution was flicked out and the wellsincubated with 100 μL of PBS containing 1% dry milk powder (w/v) for onehour in order to block the surface for unspecific binding. The procedurecould be applied also to sulphatide bound to biotinylated albumin.

Assay Protocol

Serum samples, 50 μL of serum diluted 1:400 with PBS containing 1% milkpowder were added to the wells of a microtiter plate treated asdescribed above. In case of high sulphatide titer sera, the samples werefurther diluted. The plates were then incubated at +4° C. over night. Apool of sera from blood donors (50 serum samples each individually foundto be negative for anti-sulphatide reactivity analysed with TLC-ELISA)was used as reference. A positive serum was used as an internalstandard. Each sample was analysed in triplicate. After incubation thesamples were flicked out and the wells washed with NUNC immunowasher 6times with 0.1% albumin dissolved in PBS. Thereafter 50 μL phosphataseconjugated anti-human IgG (Zymed, BioZac, Sweden) diluted 1:500 with 1%albumin in PBS were added and the plate incubated for 1 hour at roomtemperature. Unbound components was then removed by washing 5 times withPBS containing albumin (1%). Thereafter incubation was performed at +37°C. with 100 μL p-nitro phenyl phosphate (1 mg/mL) (phosphatase-substratetablets 104, Sigma, U.S.A.) dissolved in 1.0 M diethanol amine buffer pH9.8. The enzymatic reaction was stopped by adding 50 μL 3 M NaOH. Theabsorbance was the read at 405 nm.

The results on clinical samples have indicated safer diagnoses in thecontext of IDDM, and have also showed, for the first time, that elevatedlevels of anti-sulphatide antibody functions as a marker for preforms ofIDDM. In addition the usefulness of anti-sulphatide antibody as a markerfor monitoring treatment regimens, the results suggesteddiagnoses/prediction of diabetic late complications with this antibodyas a marker.

We claim:
 1. A method for assaying for an anti-glycolipid antibody comprising contacting a sample containing the anti-glycolipid antibody with a complex of a water soluble polymer containing hydrophobic regions and the glycolipid.
 2. The immunoassay method of claim 1, wherein the polymer and the glycolipid are non-covalently bound to each other.
 3. The immunoassay method of claim 1, wherein the water-soluble polymer is a a delipidized protein that in native form binds lipids.
 4. The immunoassay method of claim 1, wherein the glycolipid is a glycosphingolipid.
 5. The immunoassay method of claim 1, wherein the glycolipid contains one mono- or disaccharide unit.
 6. The immunoassay method of claim 1, wherein the glycolipid is sulphatide.
 7. The immunoassay method of claim 1, wherein the water-soluble polymer is albumin.
 8. A method for assaying for a glycolipid, comprising contacting a sample containing the glycolipid with anti-glycolipid antibodies and with a complex of a water soluble polymer containing hydrophobic regions and the glycolipid.
 9. The immunoassay method of claim 8, wherein the polymer and glycolipid are non-covalently bound to each other.
 10. The immunoassay method of claim 8, wherein the water-soluble polymer is a delipidized protein that in native form binds lipids.
 11. The immunoassay method of claim 8, wherein the glycolipid is a glycosphingolipid.
 12. The immunoassay method of claim 8, wherein the glycolipid contains one mono- or disaccharide unit.
 13. The immunoassay method of claim 8, wherein the glycolipid is sulphatide.
 14. The immunoassay method of claim 8, wherein the water-soluble polymer is albumin.
 15. A method of diagnosis of neuropathy or insulin-dependent diabetes mellitus (IDDM), comprising assaying for anti-sulphatide antibody by contacting a sample with a complex of a water-soluble polymer containing hydrophobic regions and sulphatide, wherein presence of anti-sulphatide antibody indicates neuropathy or insulin-dependent diabetes mellitus.
 16. Method of diagnosing prediabetic forms of insulin-dependent diabetes mellitus (IDDM) in an individual that does not exhibit clinical symptoms of IDDM, comprising assaying for anti-sulphatide antibody in a sample with a complex of a water soluble polymer containing hydrophobic regions and sulphatide, wherein a detected elevated level of the antibody compared to the healthy population is taken as a marker for a prediabetic state of the individual. 